The overall goal of this research is to obtain significant structural information on the Alpha and Beta chains of human I-E (DR) and I-A antigens. This information will be used to (a) compare the sequence homology of these antigens to each other and to immunoglobulins, (b) enable Dr. Keiichi Itakura (in a separate proposal) to construct mixed DNA-probes which will be used in the molecular cloning of these respective cDNAs and genes, and (c) verify the nature and structure of the antigens for which DNA sequences are obtained. Ia antigens are purified from cell-lines by combined lentil-lectin affinity chromatography and monoclonal-immunosorbent chromatography followed by preparative SDS-gel electrophoresis. We have obtained extended NH2-terminal sequences with approximately 1 mole samples of the Alpha chains of human I-E and I-A antigens and the Beta chain of I-E. The Beta chains of I-A are presently obtained as mixtures of I-A and I-E Beta chains, but will be later separated by reverse-phase high performance liquid chromatographyy (RPLC) for structural analysis. The approach for primary sequence analysis includes preparation of tryptic peptides of purified chains, separation by RPLC, and microsequence analysis. Alignment of tryptic fragments will be performed by comparison to cDNA sequences obtained by Drs. Keiitchi Itakura and Jack Silver. These studies will ultimately enable us to perform a detailed structural analysis of Ia antigens in MHC-linked diseases, and possibly lead to the development of a diagnostic test for these diseases, and an understanding of the disease mechanisms at the molecular level.